Apparatus for the detection of living microbial contaminants in petroleum products



- April 11, 1967 M. E. GEORGE 33%,712 APPARATUS FOR THE DETECTION OF LIVING MICROBIAL CONTAMINANTS IN PETROLEUM PRODUCTS Filed March 25, 1965 IN VENTOR.

United States Patent Office 3,313,712 Patented Apr. 11, 1967 3,313,712 APPARATUS FOR THE DETECTION OF LIVING MICROBIAL CONTAMINANTS IN PETROLE- UM PRODUCTS Marilyn E. George, 1211 Far Hills Ave., Dayton, Ohio 45419 Filed Mar. 25, 1963, Ser. No. 267,866 2 Claims. (Cl. 195-127) The invention described herein may be manufactured and used by or for the Government for governmental purposes without the payment to me of any royalty thereon.

This invention relates to the detection of living microbiological growth or contamination in petroleum products such as fuel and oil. Such contamination is particularly onerous in connection with jet aircraft because of the vulnerability of jet aircraft fuel systems to contaminated fuel, and because of the control problem of fuel associated with world wide operations far removed from the domestic sources of such fuel and oil. While not limited to such product, the invention will be disclosed in connection with JP-4 fuel, which is representative of both petroleum products in general, and to the application of such products.

Microorganisms have been found actively growing in fuel storage tanks, in integral wing tanks of jet aircraft, and in components of fuel-handling systems. There is strong evidence that microorganisms and/or their metabolic by-products are implicated in the clogging of fuel filters, corrosion of fuel system materials, and actual changes in fuel composition. Adding to the complexity of the problem is the wide variety of types of organisms which have been found growing in fuel samples. These include some common aerobic bacteria, sulfate-reducing bacteria, iron depositing bacteria, strict and facultative anaerobic bacteria, several genera of filamentous fungi, yeasts and other microorganisms which cannot be classified.

One of the difficult aspects in the study and control of these microorganisms is the detection of the living cells in fuel before the appearance of any physical results of their metabolic activity; such as sludge or slime forma tion. Another problem is the differentiation of living cells from dead cells, and from other organic and inorganic contaminants. It is not practical or advantageous to use standard biological methods for enumerating or detecting organisms, or for growth studies, without either altering the metabolic patterns or actually killing the organisms.

This invention provides a means for use in the detection of a metabolic by-product of living cells. All living micro organisms produce gases, such as carbon dioxide, as a product of metabolism. This invention provides a means for use in the detection of the carbon dioxide produced during cellular respiration of any organism contained in the fuel sample undergoing test. Nonviable cells, other organic and inorganic contaminants, and atmospheric carbon dioxide levels do not adversely affect the detection process or results.

The primary object of this invention is to provide a means for detecting metabolism of living microorganisms in petroleum products.

Another object of this invention is to provide a means for detecting microbial growth before there is any visual evidence of such growth in the test speciman itself.

Yet another object of this invention is to provide a detection apparatus made of components having negligible value and readily available in any laboratory or drug store.

A still further object of this invention is to provide an apparatus, which may be used by a layman, for detecting microbial activity.

Still another object of this invention is to provide a means for detecting microbial activity which requires only visual observation.

A preferred embodiment of the invention is shown on the accompanying drawing on which the test apparatus is referred to as apparatus 10. The apparatus comprises a sample holding container 12 having an opening which may be closed by a removable closure means or stopper such as cork 14. Extending from the lower face of cork 14 is a hollow gas permeable container means such as transparent open end tube 16 shown retained by axially passing through the cork. The container 12 and tube 16 may be made from any material which is chemically inert to the product being tested; however, they are preferably made of glass in order that both the tube 16 and the test specimen may readily be observed from the outside.

The petroleum or water bottom specimen to be tested is placed within container 12. The specimen shown on the drawing consists of the upper and lighter specific gravity J P-4 fuel 18 and the lower and heavier specific gravity water bottom or mineral salts medium 20, with interface 22 between the two liquids. The water bottom 20 comprises the solution usually accumulating at the bottom of fuel storage tanks or in the sump areas of fuel handling systems and consists of water condensate, rust and other contaminants accumulating in the normal course of events. This water bottom is often discolored to visually appear as microbiologically contaminated fuel; although in reality, no such contamination is present.

The test specimen may consist of a single solution. An example using a two-liquid test specimen is being shown since the problem is more complex than the single solution problem, in that the cause of the discoloration of the lower liquid 20 is not known.

It was found highly successful to make tube 16 from a glass tube having an internal diameter of 1.85 mm. and a len th of mm. The tube should be packed with a colorimetric indicator sensitive to one or more of the gases of the metabolic byproduct. Indicating soda lime, 200 mesh, as manufactured by the Mallinckrodt Company produced excellent results. The lower end of the glass tube should be loosely plugged with a material such as cotton batting or tissue paper 1612 which is chemically inert to carbon dioxide gas and which is gas permeable to permit passage of the gaseous by-product to the colorimetric indicator. The tube is suspended approximately 5 mm. above the fuel air interface 24. Paraflin wax 26 or a sealing Wax may be used to seal the potential entrances into the container; thus preventing the ingress of carbon dioxide from the atmosphere. The outer end of tube 16 may be sealed if desired; however, it has been found unnecessary to do so because the small proportion of carbon dioxide in the atmosphere acting on the indicating soda lime at the upper end of the tube has no effect on the soda lime at the lower end of the tube.

If there is any microbial activity in the test specimen, such as microbial growth 28 shown on interface 22, the released carbon dioxide gas permeates into the lower end of tube 16 and progressively changes the color of the indicating soda lime from its original pink to a yellowish color. The color change commences at the lower end of the tube and progresses upwardly while maintaining a line of demarcation or a diffusion Zone between colors as indicated by line 30 on the drawing.

The procedure a'bove disclosed has proved itself successful for the qualitative detection of microorganisms in fuel and water bottoms. The samples with no active microbial contamination have remained consistently negative, and those with active microbial contamination have shown color change in the indicating soda lime in varying amounts. The length of a test period cannot be firmly stated because of the many variables involved. Such variables include, for example: the size of container, the volume of the test specimen, the ambient temperature, the level and activity of microbial contamination, and the configuration of tube 16.

If desired, tube 16 may be omitted and a nongranular colorimetric indicator may be extended from the closure means 14. Such a colorimetric indicator could be formed by using suitable binders or other means of forming a self-supporting indicator. Likewise, substances other than indicating soda lime, which are sensitive to carbon-dioxide gas, may be used as a colorimetric indicator to detect one of the other gaseous by-products of the metabolic process of living microorganisms.

It is to be understood that the embodiment of the present invention as shown and desoribed is to be regarded as illustrative only, and that the invention is susceptible to variations, modifications and changes within the scope of the appended claims.

I claim:

1. An apparatus for detecting the presence of living microbial growth in a liquid petroleum-water mixture and which growth is capable of liberating carbon dioxide as a metabolic by-product comprising a transparent container for holding said petroleum-water mixture and having an opening therein,

removable means closing the opening in said container,

a transparent hollow tube open at its upper and lower ends and which is retained by and passes through said closure means with the lower end of said tube extending downward within said container so as to be above the liquid petroleum-water mixture and with the upper end of said tube open to the atmosphere,

a colorimetric chemical indicator within said hollow tube and sensitive to the carbon dioxide liberated by said living microbial growth, and

a gas permeable plug inserted in the lower open end of said tube to retain the chemical indicator therein.

2. The apparatus of claim 1 wherein the indicator is soda lime.

References Cited by the Examiner UNITED STATES PATENTS 1,512,893 10/1924 Fulweiler 195-103.5 2,294,425 9/1942 Sanderson 195--103.5 2,940,834 6/1960 Ringrose 23254 2,990,339 6/1961 Frank et al. 195139 3,014,848 12/1961 Ferrari 195103.5 3,033,761 5/1962 Brown 195103.5 3,038,841 6/1962 Swartwout 195139 3,047,370 7/1962 Avtges et a1 23252 3,058,813 10/ 1962 Barney et a1 23253 3,068,073 12/1962 Stanford 23232 OTHER REFERENCES Beerstecher, Petroleum Microbiology, Elsevier Press Inc., New York, 1954, pages 155 to 160.

A. LOUIS MONACELL, Primary Examiner.

A. E. TANENHOLTZ, Assistant Examiner. 

1. AN APPARATUS FOR DETECTING THE PRESENCE OF LIVING MICROBIAL GROWTH IN A LIQUID PETROLEUM-WATER MIXTURE AND WHICH GROWTH IS CAPABLE OF LIBERATING CARBON DIOXIDE AS A METABOLIC BY-PRODUCT COMPRISING A TRANSPARENT CONTAINER FOR HOLDING SAID PETROLEUM-WATER MIXTURE AND HAVING AN OPENING THEREIN, REMOVABLE MEANS CLOSING THE OPENING IN SAID CONTAINER, A TRANSPARENT HOLLOW TUBE OPEN AT ITS UPPER AND LOWER ENDS AND WHICH IS RETAINED BY AND PASSES THROUGH SAID CLOSURE MEANS WITH THE LOWER END OF SAID TUBE EXTENDING DOWNWARD WITHIN SAID CONTAINER SO AS TO BE ABOVE THE LIQUID PETROLEUM-WATER MIXTURE AND WITH THE UPPER END OF SAID TUBE OPEN TO THE ATMOSPHERE, A COLORIMETRIC CHEMICAL INDICATOR WITHIN SAID HOLLOW TUBE AND SENSITIVE TO THE CARBON DIOXIDE LIBERATED BY SAID LIVING MICROBIAL GROWTH, AND A GAS PERMEABLE PLUG INSERTED IN THE LOWER OPEN END OF SAID TUBE TO RETAIN THE CHEMICAL INDICATOR THEREIN. 